The present invention relates to the separation of a compound or compounds of interest from a mixture or mixtures using high performance liquid chromatography.
Various methods for identifying and separating reaction products from mixtures are known. These methods are important to the formation of purified libraries of compounds produced by combinatorial chemistry.
U.S. Pat. No. 5,670,054 teaches an automated high performance liquid chromatography (HPLC) system that separates, identifies, purifies, and quantitates complex mixtures of reaction products or natural products on a semi-preparative or preparative scale. The patent teaches generating and digitally storing a chromatogram from a first HPLC column along with mass spectrometric data to guide sample purification on a semi-preparative or preparative HPLC system.
There is a need for a method of separating reaction products from mixtures that does not rely on electronically stored UV chromatographic data and mass spectrometric data to guide a preparative HPLC system.
The present invention relates to separating compounds of interest from mixtures using high performance liquid chromatography (HPLC). According to one aspect of the present invention, a method of separating a compound of interest from a mixture is provided that comprises the steps of: (a) providing a mixture containing a compound of interest, the compound of interest having an expected mass; (b) subjecting a portion of the mixture to a separation using an analytical HPLC column to produce an eluate stream; (c) analyzing the eluate stream using a mass spectrometer to determine a retention time of the compound of interest on the analytical HPLC column; (d) predicting an elution time for the compound of interest from a preparative scale HPLC column using the determined retention time of the compound of interest on the analytical HPLC column; (e) subjecting all or a portion of the remaining mixture to a preparative scale HPLC system comprising a preparative scale HPLC column; and (f) collecting at least a portion of the compound of interest using the predicted elution time.
In one embodiment, the elution time for the compound of interest is predicted by: (1) predicting a retention time of the compound of interest from the preparative scale HPLC column using a predetermined correlation function between the analytical HPLC column and the preparative scale HPLC column along with the determined retention time of the compound on the analytical HPLC column; and (2) selecting a window of time around the predicted retention time within which the compound is expected to elute. In another embodiment, a dynamic correlation function is used to predict the elution time for the compound of interest. In a further embodiment, the elution time for the compound of interest is predicted using an artificial neural network.
According to another aspect of the present invention, a method of separating compounds of interest present in a plurality of reaction product mixtures is provided. The method comprises the steps of: (a) providing a plurality of reaction product mixtures, each mixture expected to contain a compound of interest having an expected mass; (b) separately subjecting a portion of each reaction product mixture to a separation using an analytical HPLC column to produce a plurality of eluate streams; (c) analyzing each eluate stream using a mass spectrometer to verify that the eluate stream contains a compound with an expected mass and to determine a retention time of each compound with an expected mass on the analytical HPLC column; (d) predicting an elution time for each compound with an expected mass from a preparative scale HPLC column using the determined retention time for each compound with an expected mass on the analytical HPLC column; (e) separately subjecting all or a portion of each remaining mixture verified to contain a compound with the expected mass to a preparative scale HPLC system comprising a preparative scale HPLC column and a fraction collector; and (f) separately collecting at least a portion of each compound verified to be present with the fraction collector using the predicted elution time for each compound.
In one embodiment, at least two eluate streams produced in step (b) are combined for analysis in step (c) in a common mass spectrometer. The at least two eluate streams are chosen such that the expected compounds of interest in the streams have different expected masses that are distinguishable by mass spectrometry.
In another embodiment, at least two eluate streams produced in step (b) are combined for analysis in step (c) in a common mass spectrometer. At least one of the eluate streams is mechanically coded such that the expected compounds of interest in the streams are distinguishable by mass spectrometry.
According to yet another aspect of the present invention, a method of separating a compound of interest from a mixture is provided that comprises the steps of: (a) providing a mixture containing a compound of interest; (b) subjecting a portion of the mixture to a separation using thin layer chromatography to determine an Rf value for the compound of interest; (c) predicting an elution time of the compound of interest on a preparative scale HPLC column using the determined Rf value for the compound of interest; (d) subjecting all or a portion of the remaining mixture to a preparative scale HPLC system comprising a preparative scale HPLC column; and (e) collecting at least a portion of the compound of interest using the predicted elution time.